Adenoviral vector with a deletion in the E1A coding region expressing a hetorologous protein

ABSTRACT

Recombinant DNA modified by a nucleotide sequence coding for a specific polypeptide sequence whose expression is sought, this recombinant DNA being appropriate to the transformation of eucaryotic cell lines, notably human or animal, the endogenous polymerases of which are susceptible of recognizing the adenovirus promoters. The DNA according to the invention is more particularly characterized by the fact that the said insertion nucleotide sequence is placed under the direct control of the early promoter of the E1A region of the genome of adenovirus.

This a continuation of application Ser. No. 07/935,459, filed Aug. 26,1992, now abandoned, which is a continuation of application Ser. No.07/790,080, filed Nov. 13, 1991, now abandoned, which is a continuationof application Ser. No. 07/456,200, filed Dec. 20, 1989, now abandoned,which is a continuation of application Ser. No. 07/293,556, filed Jan.5, 1989, now abandoned, which is a continuation of application Ser. No,06/799,938, filed Nov. 20, 1985, now abandoned.

The invention concerns a recombinant DNA including a nucleotide sequencecoding for a specific polypeptide under the control of an adenoviruspromoter, the vectors containing this recombinant DNA, the eucaryoticcells transformed by this recombinant DNA, the excretion products ofthese transformed cells and their applications, notably to theconstitution of vaccines.

Human adenoviruses possess a long (around 36,000 bp) linear anddouble-stranded genome which codes for at least 30 proteins. The viralcycle, in the course of the infection of permissive cells, is dividedinto two phases, early and late. It is known that the four regions ofthe viral genome expressed in the early phase are called regions E1, E2,E3, and E4, whose respective positions in the whole genome areschematically represented in FIG. 1. The E1 region, situated at the leftend of the genome, is itself divided into two regions, E1A and E1B. Thepassage from the early phase to the late phase, marked by thereplication of the viral DNA, is characterized by an abrupt change inthe genetic program of the virus. The expression of certain early genesis repressed while the transcription of the late genes is accomplishedprincipally via only one promoter, the major late promoter (FIG. 1). Inaddition, a strong repression of protein synthesis of the host cell maybe observed.

The genetic organization of type 2 or 5 human adenovirus (Ad2, Ad5) issufficiently well known that their genome may be manipulated in vitroand its use as a vector for the expression of a foreign gene in ananimal cell in culture has already been envisaged. Indeed it is knownthat the E3 region, which represents 6% of the genome, is not essentialin vitro and may therefore be substituted in its entirety. The size ofthe foreign DNA fragment which it is possible to insert into the genomeof these viruses, is large. In fact, the virus may encapsulate a genomewhose length exceeds by 5% that of the wild genome.

Different vectors derived from adenoviruses of type 2 or 5 havetherefore been constructed. In these recombinants, the foreign gene wasexpressed under the control of the major late promoter. This haspermitted the obtaining in certain cases of a synthesis of the proteincoded by a foreign gene at a level comparable to that of the late viralproteins. This being the case, it results from the preceding that theexpression of the foreign gene under the control of the late promotercan only manifest itself in the late phase of the viral cycle.

The invention results from the observation that the promoter of theearly region E1A of the genome of an adenovirus (hereafter designatedsimply as “E1A promoter”) could control in a particularly effectivemanner the expression of a heterologous gene (that is, foreign vis-a-visthe genes normally associated with it in the adenovirus) or moregenerally of a heterologous nucleotide sequence coding for a polypeptidesequence whose expression is sought. In other words, the E1A promoteracts like a strong promoter, and this more particularly when the E1Apromoter combined with the heterologous coding sequence is inserted intoa viral vector.

The invention therefore concerns in a general fashion a recombinant DNAfor the transformation of eucaryotic cell lines, notably human oranimal, chosen from among which are infectable by the adenoviruses orwhose endogenous polymerases are likely to recognize the adenoviruspromoters, this recombinant DNA being, in addition, modified by aninsertion nucleic acid containing a nucleotide sequence coding for apolypeptide sequence whose expression in the said cell lines is sought.This recombinant DNA is more particularly characterized by the fact thatthe said insertion sequence is placed under the direct control of theearly promoter of the E1A region of the genome of the adenovirus.

Preferably, this recombinant DNA is incorporated into a replicatablevector in the said cell lines or associated by genetic recombinationwith such a vector.

Being a viral vector, notably one derived from adenovirus, equallyoffers the advantages attached to the E1A region of the adenoviruses,namely that its expression is constitutive and permanent all during theviral cycle.

A particularly prefered form of the recombinant DNA according to theinvention is characterized by the fact that it includes, ‘downstream’ ofthe insertion nucleic acid, in the direction of transcription, adefective adenovirus genome including nevertheless all of those of theessential sequences necessary to the replication of the correspondingadenovirus, which are normally situated ‘downstream’ of the genesnormally under the direct control of the E1A early promoter in the saidgenome.

Advantageously, the defective adenovirus genome with which therecombinant DNA conforming to the invention is associated, isconstituted of complete adenovirus genome, deprived however of theanterior part of the E1A region of the viral genome, notably of its0-2.6% fragment (the percentage expressed relative to the total size ofthe adenovirus genome).

The recombinant DNAs of the invention, associated with the elements ofvectors such as those which have been mentioned above, constitute infact the vectors will again be the case where reference is made to“defective recombinant viruses”, when the elements of the vectorsassociated with the recombinant DNA of the invention will be derivedfrom a defective genome of adenovirus. These defective recombinantviruses are advantageously used for the transformation of transformablecell lines from superior eucaryotes (notably of human or animal origin)themselves including a distinct sequence of nucleotides apt tocomplement the part of the genome of the adenovirus which is missingfrom the aforesaid vector, the said distinct sequence preferably beingincorporated into the genome of the cells of the said cell line.

As a prefered example of such cell lines, one might mention the line293, a human embryonic kidney line which contains, integrated into itsgenome, the first eleven percent of the left end of the genome of anAd5. This permits the complementing of the defective recombinant viruseswhich have deletions of this region.

The use of these systems: defective recombinant virus vector—cellscontaining a sequence capable of complementing the defective recombinantviruses, is of a very particular interest, when the nucleotide sequencecontained in the insertion nucleic acid of the recombinant DNA codes fora protein which, when it is expressed in the natural cellular host underthe control of its natural promoter, is excreted into the culture mediumof this natural cellular host.

The S gene of the genome of the virus of hepatitis B constitutes in thisregard a nucleotide sequence of particular interest, arid this forseveral reasons. On the one hand, the product of the expression of the Sgene in the cells which express it, HBsAg, is secreted into the cellularsupernatant in the form of particles which are easy to detect and toquantify by radio-immunology, which permits a precise evaluation of thecapacity of expression of the viral vector. On the other hand, theinvention provides a recombinant viral vector permitting the study ofthe expression of the genes of the HBV at the level of transcription aswell as that of translation, which is all the more interesting in thatuntil now there had not existed a cell culture system capable ofpropagating the hepatitis B virus (HBV). And lastly, the cellularinfection by the adenovirus-HBV recombinant virus illustratesparticularly well the methodological basis of a process for themanufacture of a vaccine against a given pathologic agent (in this casethe hepatitis B virus in the example under consideration). Anothernucleotide sequence of the genome of the hepatitis B virus of particularinterest is the S gene along with its pre-S2 region which codes for theHBs antigen and for a receptor of polymerized human serum albumen (pHSA)(25, 26).

It goes without saying that one may substitute for the S gene into therecombinant DNA, any other nucleotide sequence coding for a distinctantigen-protector against another given pathogenic agent, especiallywhen this distinct antigen-protector is itself normally susceptible ofbeing secreted by the cells transformed by the recombinant DNA. Itequally goes without saying that in the recombinant DNA the S gene andthe pre-S2 region may be replaced any other nucleotide sequence codingfor a distinct antigen-protector against another given pathogenic agent,especially when this distinct antigen-protector is itself normallysusceptible of being secreted by the cells transformed by therecombinant DNA. The nucleotide sequence coding for this distinctantigen-protector may possibly be inserted into the recombinant DNA inphase with another genes for example the HBsAg antigen, if and when thatother gene may be used as the “locomotive” for the promotion of theexcretion equally of this distinct antigen, notably in the form of ahybrid protein. As an example of the distinct antigens susceptible ofbeing thus produced (if need be, in the hybrid protein form), thestructural glyco-proteins of the Epsteim-Barr virus may be mentioned.

The first nucleotides of the nucleotide sequence coding for a specificpolypeptide (a “simple” or hybrid protein) are placed, notably byconstruction, as close as possible to this promoter, notably to the“TATA box”, which is characteristic of the promoter, it being understoodhowever that the nucleotide sequence between the promoter and the ATGinitiator of the nucleotide sequence coding for the said specificpolypeptide should in general contain the triplets coding for thenon-translated 5′ end of the messenger RNA corresponding normally to thecoding sequence and containing the matching sequences to the ribosomesnecessary to an effective translation. This untranslated 5′ end of themessenger RNA could also be replaced by the untranslated 5′ end of amessenger RNA distinct from that normally associated with a specificcoding sequence. For example, one may, in the case of the S gene,replace the untranslated 5′ end containing the pre-S gene or juxtaposingthis with the untranslated 5′ end of the messenger RNA of the T antigenof SV40. But it has also been noted that when using a DNA sequencecontaining the o and pre-S2 regions of the genome of the hepatitis Bvirus under the control of the strong promoter, E1A, it is possible toobtain the expressions both of the pre-S2 and the S regions. Any otheruntranslated 5′ end of messenger RNA may be used if it is compatibleSmith the other similar chosen end.

It is advantageous that the distance between the TATA box of thepromoter and the initiation site of the messenger RNA should be around30 nucleotides.

The E1A promoter of the recombinant DNA according to the invention andmore generally the vector according to the invention using the moreimportant parts of the adenovirus genome are preferably derived from anadenovirus belonging to category C, as defined by TOOZE. Theseadenoviruses have the known property of not being oncogenic. Thesub-types Ad2 or Ad5 of this category of adenovirus are characterized byan important transforming power. The use of this latter type ofrecombinant DNA is therefore particularly recommended, when the desiredexpression product is intended for the production of antigen-protectors,notably the active principles of vaccines. This will be even more truein the case where whole adenoviruses, and even infectious, will be usedas the active principles of live vaccines, notably under the conditionswhich will be spelled out further on.

The invention naturally equally concerns the cell lines, notably ofhuman or animal origin, which are transformed by the recombinant DNAs asdefined hereabove and which are rendered capable of synthesizing apolypeptide coded by the nucleotide sequence (or the said nucleotidesequences) contained in these recombinant DNAs and placed under thedirect control of said promoter.

The invention concerns more particularly yet the cell lines transformedwith the recombinant DNA conforming to the invention and in additioncharacterized in that the cells of the cell lines themselves contain adistinct sequence of nucleotides capable of complementing the part ofthe adenovirus genome which is missing from the aforesaid vector, thesaid distinct sequence being preferably incorporated into the genome ofthe cells of the said cell line.

In this regards the line 293 already mentioned above, after having beentransformed by the recombinant DNAs, constitutes a prefered cellularculture according to the invention. Due to the complementation sequencecontained by the cells of this line, a major viral multiplication withinthese cells is observed and, by way of consequence, an equallymultiplied expression of the sequence coding for the predeterminedpolypeptide. In the first case where this coding sequence is the S gene,a high production is obtained of the HBsAg antigens excreted into theculture medium of these cells. In the second case where the codingsequences are constituted of the S gene and the pre-S2 region of thehepatitis B virus, the recombinant adenovirus directs in vitro thesynthesis of the HBsAg particles possessing an activity for the pHSAreceptor. Injected into rabbits this recombinant virus producesanti-HBsAg and anti-pHSA antibodies. This shows the possibility of usingthe recombinant adenovirus to express a gene both in vitro and in vivo.The same vectors may be used for the transformation of Vero cells underanalogous conditions.

The vectors containing the recombinant DNA according to the inventionmay equally be used for the transformation of cells not possessingthemselves the complementation sequence under the conditions which havebeen indicated hereabove. It may then be necessary to proceed to aco-transformation of these latter types of cells, on the one hand, witha vector containing the recombinant DNA according to the invention, andon the other, with a non-defective adenovirus or a distinct recombinantDNA containing the adenovirus sequences which are missing from therecombinant vector conforming to the invention. It may certainly beobserved in this latter case a simultaneous production of HBsAg antigens(when the coding sequence contains the S gene) arid of the adenovirusreplicated and liberated by the cells thus transformed. Theantigen-protector formed may however be separated from the viralsuspension, if need be, for example by bringing the culture medium intocontact with anti-adenovirus antibodies, preferably immobilized on asolid support, such as reticulated agarose, sold under the designation,SEPHAROSE. In any case, the presence of residual quantities of the virusin the vaccinating preparation is only of a relatively minor importance.Indeed, the adenovirus has only a weak pathogenicity in humans, causingno more than benign respiratory infections.

The small importance of the pathogenicity of the adenoviruses, moreparticularly of those belonging to group C of the human adenoviruses,allows the contemplation of “living vaccines”. These could beconstituted of infectious adenoviruses modified at the level of the E3region by the insertion of the recombinant DNA according to theinvention in the non-essential part of the adenovirus. In this regard,the fact must be emphasized that the human adenoviruses of the C grouphave never proved to be tumorigenic in animals (3). These vectors orviruses will be of a very particular interest for the transformation ofVero cells, the non-tumorigenic character of which has been firmlyestablished. For this reason they constitute a line of animal originparticularly favorable to the production of products for human use.

The supplementary characteristics of the invention will appear yet inthe course of the description which follows of the preferedconstructions of the vectors containing the recombinant DNA according tothe invention and of the conditions under which these vectors areuseable. For this, reference will be made to the drawings, in which:

FIG. 1 schematically represents the relative positions of the differentregions of the genome of an adenovirus of sub-type Ad5, and, at a largerscale, of the E1A region of this genome;

FIG. 2 is a now classic diagram of the genome of the virus of hepatitisB;

FIGS. 3 to 6 show the successive constructions leading to therealization of a plasmid containing a first recombinant DNA conformingto the invention (FIG. 6); and

FIG. 3 schematically represents the construction of the pE1a plasmid andrelevant restriction sites.

FIG. 4 schematically represents the construction of the pAB1 plasmid andrelevant restriction sites.

FIG. 5 schematically represents the construction of the pK4 plasmid andrelevant restriction sites.

FIG. 6 schematically represents the construction of the pK4(S) plasmidand relevant restriction sites.

FIG. 7 schematically represents the construction of a “defectiverecombinant virus” starting from the modified plasmid of FIG. 5 and thedefective genomes of Ad5.

First, the following observations will be made concerning the figures,before describing the realization of the construct ion of therecombinant DNAs according to the invention.

In FIGS. 3 to 6, the parts drawn with a fine line correspond tosequences of the plasmid pML2.

The numbers appearing in the FIGS. 1 to 5 indicate the positions of therestriction sites in the viral sequences: Ad5, SV40, and HBV. Thenumbering of the positions of the restriction sites for Ad5 and SV40 isthat of J. TOOZE and for HBV is that of P. TIOLLAIS et al.

The site designations which have been crossed out in the drawings bearwitness to the former presence of these same sites in the correspondingparts of the DNAs which will be described hereafter. These sites havemeanwhile been deleted, suppressed by repairing the cohesive ends of thefragments opened or fragmented with the aid of the correspondingrestriction enzymes, or by any other means such as envisaged in thedescription which follows of the constructions which have been made.

1. Reminder of the Principle Elements of the Structure and of theOrganization of the Genome of the Adenovirus Ad5 (Hereafter Often SimplyDesignated as Ad5)

They result in the parts 1A and 1B of FIG. 1.

1A: The genome is linear, double-stranded molecule of DNA of around36,000 bp. The arrows indicate the position and the direction of thetranscriptions of the early regions E1A, E1B, E2, E3, and E4. The majorlate promoter, PM_(t) (“promoteur majeur tardif”), and the transcriptionunit associated with it are equally shown. The numbering, by tens, from0 to 100, corresponds to the sizes expressed as a % of the length of thetotal genome.

1B: The region E1A. The transcripts of this region all have identical5′P (position 499) and 3′OH (position 1632) ends. The first T of theTATE element of the promoter is situated at position 468. Therestriction sites used in the plasmid constructions are indicated. Thesizes are expressed by the number of base pairs.

2. Origin of the Fragments Containing the S Gene or the S Gene and thepre-S2 Region Utilized in the Constructions which Follow

First case: The fragment of the genome of the hepatitis B viruscontaining the S gene.

It is derived from the genome of the virus of hepatitis B (FIG. 2). Itis to be recalled that the genome of the hepatitis B virus is a circularmolecule of DNA partially single-stranded. Its length is around 3,200dp. It is constituted by the pairing of two strands of unequal lengthcalled strands L(−) and S(+). The S gene represents the coding sequenceof the major polypeptide of the viral envelope of the HBsAg. The DNAfragment used in the constructions below is the XhoI₁₂₇-BglII₁₉₈₄fragment. The polyadenylation site of the HBsAg has been localized atposition 1916.

Second case: The fragment of the genome of the hepatitis B viruscontaining the S gene and the pre-S2 region.

The DNA fragment utilized is the MstII₃₁₆₁-BglII₁₉₈₂ which codes bothfor the HBs antigen and for a receptor of polymerized human serumalbumin (pHSA). The MstII site precedes the initiation codon of thepre-S2 region of 9 nucleotides. The BglII is situated 64 nucleotidesfurther on from the poly A addition signal of the S gene.

It is a question in what follows of the construction and of thepropagation of a recombinant adenovirus which includes the S gene. Theoperating method is identical to obtain a recombinant adenovirus,conforming to the invention, possessing the S gene and the pre-S2region, it being understood that, in the second case, it is theMstII-BglII fragment of DNA which is inserted between the HindIII andBamHI restriction sites of the plasmid pK4, in place of the XhoI-BqlIIDNA fragment discussed below.

In what follows, the recombinant adenovirus possessing the S gene willbe designated as Ad5(X-B) and the recombinant adenovirus possessing theS gene and the pre-S2 region as Ad5(M-B).

3. Construction of the Plasmid pE1A (TaqI) (FIG. 3)

The plasmid pE1A(TaqI) contains the first 632 nucleotides of the leftend of the Ad5 genome. This fragment has been obtained by cutting of thepurified SacI E restriction fragment (0-5.0%) of Ad5 by TaqI. (FIG. 1).This fragment has been inserted between the EcoRI and ClaI restrictionsites of the pML2 plasmid (FIG. 3). The pML2 plasmid has been opened byEcoRI and ClaI. The Ad5 fragment has been bound to the linearizedplasmid at the level of the TaqI end. The junction of the TaqI-ClaI endsre-creates a ClaI restriction site. The EcoRI end of the recombinant hasbeen repaired with the DNA-polymerase I of E. coli (Klenow's fragment)and the plasmid re-circularized by means of the ligase T4. The EcoRIsite has therefore been reconstituted.

4. Fabrication of the Plasmid pAB1 (FIG. 4)

The pAB1 plasmid has been constructed from the pEIA(TagI) plasmid, in away so as to eliminate the coding part of the E1A region. This has beencarried out by isolation of the PvuII-PvuII fragment (positions452-623), cutting of this fragment by the enyme HaeIII (position 495),and reinsertion of the Pvu₄₅₂-Hae₄₉₅ fragment at the level of thePvuII₆₂₃ site of the pE1A(TaqI) plasmid. In other words, theHaeIII₄₉₅-PvuII₆₂₃ fragment has been deleted. The pAB1 plasmid containsa HindIII site close to the transcription initiation site of the E1Aregion and a BamHI site (of pML2) situated at a distance. These tworestriction sites may be used to clone foreign genes without geneticfusion.

5. Production of the Plasmid pK4 (FIG. 5)

pAB1 has been cut by HindIII and BamHI and the fragment containing theE1A promoter, issued from pAB1, has been linked, at the level of itsBamHI end to the BqlI-BamHI fragment (positions 5235-2533 of the genomeof the SV40 virus) hereafter called A(SV40) containing the gene coding,for the T and t antigens of the SV40 virus. After repairing of the BglIand HindIII ends of the reconstituant by Klenow's fragment, the plasmidis re-circularized by means of the liqase T4. The present constructionin the pK4 plasmid has been tested by bringing into play the transitoryexpression of the T gene. Introduced into HeLa cells by transfectionaccording to the calcium phosphate technique the pK4 plasmid directs thesynthesis of the T antigen of SV40 which has been detected byimmunofluorescence. Around 1% of the transfected cells presented a clearfluorescence. The absence of fluorescence after cellular transfection bya plasmid containing the fragment of SV40 inserted in the wrongorientation shows that the gene of the T and t antigens is well placedunder the control of the E1A promoter of the Ad5.

It is the HindIII site, at position 5171 of the A(SV40) fragment whichis then used to substitute the aforesaid fragment containing the S genefor the major part of A(SV40).

6. Production of the Plasmid pK4S(X-B) (FIG. 6)

The pK4 plasmid has been digested by HindIII and BamHI. The XhoI-BglIIfragment (positions 125 to 1982) of the genome of the hepatitis B virus(HBV) (FIG. 2) has been inserted, instead and in the place of the majorpart of A(SV40) between the HindIII and BamHI restriction sites of thepK4 plasmid, after repairing of their respective ends by the DNApolymerase I of E. coli (Klenow's fragment) (FIG. 3). The XhoI, HindIII,BamHI and BglII restriction sites are lost after ligation.

The HindIII site was situated at 8 nucleotides before the ATG initiatorof the T and t antigens. The insertion of the S gene into this sitetherefore permits the conservation of the 5′ end of the early mRNA ofSV40 containing the “capping” site and the pairing sequences of themessenger to the ribosomes. The HBV DNA fragment contains the codingsequence or S gene (position 155 to 833) of the major polypeptide of theviral envelope bearer of the HBsAg as well as the sequence situated in3′ of the S gene and which includes the polyadenylation site off themessenger RNA of the HBsAg at position 1916. Two plasmids, pK4S⁺ andpK4S⁻, bearers of the HBV fragment inserted in both directions have beenisolated. 293 cells have been transfected by these two plasmids and thesynthesis of HbsAg was sought in the cellular supernatant 3 days afterthe transfection. Only the pK4S⁺ plasmid which possesses the E1Apromoter at the 5′ end of the S gene is capable of directing thesynthesis of HbsAg. This shows that the expression of the S gene isindeed under the control of the E1A promoter of Ad5. Finally, anon-methylated ClaI restriction site in E. coli has been introduced intothe plasmid pK4S⁺ (X-B) at the level of the NruI site of the pML₂sequence. The site is necessary to the construction of the recombinantvirus.

It is finally the fragment delimited by the PstI and ClaI ends, andobtained from pK4S(X-7B), which has been used for the fabrication of a“defective recombinant virus” conforming to the invention.

7. Construction of the Defective Recombinant Virus (FIG. 7)

3 micrograms of the PstI-ClaI restriction fragment purified from thepK4S⁺ (X-B) plasmid have been ligated to 20 micrograms of the ClaIrestriction fragment (2.6%-100%) of the Ad5 purified byultracentrifugation in a sucrose gradient to provide the “defectiverecombinant virus” Ad5.

8. Propagation of the Recombinant Virus

The 293 cells have been cultured in dishes 6 cm in diameter. 4 hoursbefore transfection the supernatant of the culture has been replaced bymedium. 5 dishes of 293 cells at 70% confluence were then trarisfectedwith the ligation mixture according to the calcium phosphate techniquethen incubated for 4 hours at 37 degrees C. After adsorption, the cellscontained in each dish have been washed with 2 ml of TS buffer (NaCl8000.0 mg/l, KCl 380.0 mg/l, Na₂HPO₄ 100.0 mg/l, CaCl₂ 100.0 mg/l,MgCl₂,6H₂O 100.0 mg/l, Tris 3000.0 mg/l, pH 7.4), treated with 400microliters of a TS solution containing 20% glycerol for 1 minute atambient temperature, washed twice with 2 ml of TS buffer, thenre-covered with 4 ml of MEM medium containing 1% noble agar, and 1%fetal calf serum. At days 4 and 7, the cells have been re-covered with 4ml of nutrient mix. At day 10, the cells have been stained with 4 ml ofnutrient medium supplemented with 0.01% neutral red. The plagues havebeen observed on day 11. The virus have been resuspended in 1 ml of TSand amplified on 293 cells. The presence of HBsAg in the culture mediumhas been tested by RIA (“radio-immunoassay”)(Austria II, ABBOTTLaboratories). After amplification, the presence of HBV sequences in therecombinant has been tested by hybridization. Five plagues have beenanalyzed. Only one contained a recombinant HBsAg⁺ virus. This Ad5(X-B)clone along with another clone were positive for the detection of theHBV sequences. The size of the recombinant viral genome exceeds that ofthe wild virus of 2100 bp. No deletions could be detected by analysis ofthe restriction fragments of the genome. In addition, this analysis hasshown that the PML2 sequences situated between the PstI restriction siteand the Ad5 sequence have been correctly excised during the propagationof the recombinant nenome in the 293 line.

9. The Synthesis of HBsAg Directed by the Ad5(X-B)

The 293 cells and Vero cells have been infected with Ad5(X-B) virus. Thelevels of expression of HBsAg synthesized are shown in Table I. Samplesof the cellular supernatant have been taken 3 days after infection andthe HBsAg sought by RIA. The results show that the Ad5(X-B) vector iscapable of directing the synthesis of HBsAg in the two cell lines, andthe excretion of HBsAg by the cell lines into their respective culturemediums.

The synthesized HBsAg has been purified by ultracentrifugation in CsCl.It has a density of 1.20. The typical particles of 22 nm have beenobserved under electron microscopy.

10. Synthesis of HBsAg Directed by Ad5(M-B) Vectors

The levels of expression of HBsAg synthesis after infection of 293 andVero cells by Ad5(M-B) are shown in Table I.

The extra- and intra-cellular distribution kinetics of HbsAg startingfrom Vero cells infected by Ad5(M-B) have indicated that the synthesisof HBsAg commenced 3 hours after infection and could be detected in themedium after 8 hours. The infection by the recombinant virus Ad5(M-B)has led to an accumulation of HBsAg of 4.5 to 1 microgram/10⁶ cells inthe medium after 120 hours. Repeated exeriments have shown that therecombinant virus containing the pre-S2 region synthesizes greaterquantities of HBsAg than the recombinant virus containing only the Sgene. The HBsAg purified from the culturemedium of cells infected withthe recombinant adenovirus Ad5(M-B) consisted of a homogenous populationof particles having a mean diameter of 22 nm. The density aftercentrifugation in CsCl was 1.21.

11. Activity of the pHSA Receptor of the HBsAg Particles Issued fromVero Cells

The HBsAg particles produced in the Vero cells have been tested by thetechnique of hemagglutination of sheep red blood cells coated with pHSAand by RIA to detect an activity of the receptor for the pHSA. A bindingactivity for pHSA has been detected, but not for polymerized bovinealbumin (Table II). No such activity could be detected with Vero cellsinfected by the recombinant adenovirus Ad5(X-B) containing only the Sgene.

12. In Vivo Activity of the Recombinant Virus

Rabbits have been intravenously inoculated with highly purifiedpreparations of Ad5(M-B) recombinant adenovirus and of wild adenovirus.Although the HBsAg antigen could not be detected in their serum, 5rabbits out of 8, inoculated with the recombinant virus, showed theappearance of an anti-HBs titer varying from 20 to 270 mIU/ml after 15days (Table III). No anti-HBs antibodies were detected in the rabbitsinjected with the wild type adenovirus. After a second intravenousinoculation 4 weeks after the first, a second peak attaining 440 mIU/mlfor one of the animals was observed in the anti-HBs response. 4 weeksafter the second injection, the anti-HBs titers varied from 6 to 360mIU/ml. Prior studies have indicated that the minimum anti-HBs levelstill offering protection against HBV in humans is 10 mIU/ml. Anti-pHSAantibodies were sought in the inoculated rabbits in order to determinetheir relationship to the neutralization of the HBV. These antibodies,detected by the inhibition of hemagglutination, have been found in 5animals out of 5 having a positive anti-HBs response (Table IV).

The recombinant adenovirus Ad5(M-B) therefore directs in vivo thesynthesis of HBsAg particles having the character of a receptor forpolymerized human serum albumin.

The invention therefore furnishes a methodological basis for thefabrication of a vaccine against hepatitis B (or against other types ofdisease) in cell cultures.

The value of using adenoviruses of type 5 as a vector is two-fold. Onthe one hand, the pathogenicity of this virus in humans is very low,causing only mild respiratory infections. On the other, this serotypewhich belongs to group C of the human adenoviruses is not tumorigenic inanimals. In addition, the rate of HBsAg production obtained on Verocells (around 1 microgram/10⁶/infectious cycle) is, a priori, the mostfavorable to the production of a product for human use.

As it goes without saying and as it results anyway from the preceding,the invention is in no way limited to the modes of application and ofrealization which have been especially outlined here; but on thecontrary it embraces all variants; among these variants, it isappropriate to mention the other forms of vectors containing therecombinant DNA according to the invention, notably the plasmids. Thesemay be, either used to create viral vectors of the kind which have beendescribed, or may themselves be used as vehicles for the incorporationof the recombinant DNA into the genome of the cells of superioreucaryotes, notably cell cultures of humans or primates.

Equally it goes without saying that for the 293 cells mentioned above,may be substituted any other higher eucaryotic cells infectable byadenoviruses or susceptible of recognizing the E1A promoter of theadenoviruses, these cells having been modified, by prior incorporationinto their own genomes, of a sequence containing the missing parts ofthe defective recombinant virus according to the invention of the genomeof an adenovirus, notably under the control of a strong promoterrecognized by these cells, for example of a promoter of thymidine kinaseor of a promoter of the SV40 virus. The sequence originating from theadenovirus, then integrated into the genome of the higher eucaryoticcells, may therefore complement the defective viruses conforming to theinvention, under conditions analogue to those permitted for the 293cells. These methods are applicable with particular advantage to Verocells.

The adenovirus used has been filed the 3rd of August 1984 under the No.I-322 with the C.N.C.M. (“Collection Nationale de Cultures deMicro-organismes”) of the INSTITUT PASTEUR of Paris.

The line 293 has been filed with C.N.C.M. the 3rd of August 1984 underthe No. I-323.

TABLE I Time course of extracellular HBsAg production Ad5(X-B) Ad5(X-B)Ad5(M-B) Ad5(M-B) infected infected infected infected Day Vero 293 Vero293 1 13 15 106 35 2 17 90 N.D. N.D. 3 45 110 349 56 4 110 100 668 60 5320 130 1057 63 6 470 120 1153 N.D. 7 550 120 900 N.D.

The cumulative amounts of HBsAg (ng) produced after infection of 293 andVero cells by either Ad5(X-B) or Ad5(M-B) are indicated.

TABLE II pHSA receptor activity of HBsAg particles from Vero cells.Human serum Cell culture supernatant HBsAg+ healthy Ad5(X-B) Ad5(M-B)Mock HBeAg+ control RIA HA RIA HA RIA HA RIA HA RIA HA pHSA 143 — 751264 119 — 10472 128 65 — pBSA  58 ND  85 ND  77 ND  145 ND 81 ND

Results are expressed in cpm for solid phase RIA and in hemagglutinationtiter (HA).

ND, not determined.

pHSA: polymerized human serumalbumin.

pBSA: polymerized bovine serumalbumin.

TABLE III Anti-HBs response (mIU ml¹) in inoculated rabbits. RabbitsWeeks 1 2 3 4 5 6 7 8 9 10 0 0 0 0 0 0  0  0 0 0 0 1 0 0 2 0 0 30  8 612 20 2 2 0 2 0 2 15 20 270 70 85 3 0 2 0 0 5 18 13 48 15 11 4 1 0 0 0 221 15 27 17 7 5 0 0 2 0 2 99 52 440 146 136 6 0 0 0 0 0 78 26 401 151148 7 0 0 0 0 0 20 27 325 58 42 8 0 0 0 0 0 20 40 367 35 6 ** (PFU =plaque forming unit).

Rabbits were injected intravenously with 10⁹ pfu of purified wild-typeAd5 (rabbits 1 and 2) or with 10⁹ pfu of purified adenovirus recombinantAd5 (M-B) (rabbits 3-10) immediately after the blood drawing in week 0and week 4. Anti-HBs antibodies have been quantitated using thecommercially available RIA Kit AUSAB from Abbott and expressed asInternational Units (3,5 RIA units are equivalent to 1 mIU).

TABLE IV Anti-pHSA receptor immune response (reciprocal of the titer)Rabbits Weeks 6 7 8 9 10 0 0 0 0 0 0 1 16 2 2 2 2 2 4 16 16 16 16 3 4 416 8 8 4 2 2 4 4 4 5 16 32 32 32 32 6 16 32 32 16 32 7 8 8 32 8 16 8 4 48 4 8

Rabbits were infected intravenously with 10⁹ pfu of purified adenovirusrecombinant Ad5(M-B) in week 0 and week 4. Single animals are identifiedby the same numbers used in Table 3.

Anti-pHSA receptor activity was expressed as the reciprocal of thehighest titer of serum able to give 100% inhibition of hemagglutination.HBsAg particles having a pHSA receptor hemagglutination titer of 1:128were mixed with an equal volume of serial dilutions of inhibitor sera.

What is claimed is:
 1. A method of transferring a nucleic acid sequenceencoding a hepatitis B virus S antigen to a mammal to induce an antibodyresponse against HBV S antigen in said mammal, comprising: administeringa defective recombinant Ad2 adenoviral vector to the mammalintravenously, wherein the vector comprises: a) an E1A promoter; b) adeletion of the entire E1A coding region; and c) a nucleic acid sequenceencoding the hepatitis B virus S antigen operatively linked to the E1Apromoter; wherein the hepatitis B virus S antigen is expressed in themammal, thereby inducing an antibody response to the hepatitis B virus Santigen in the mammal, wherein the antibody response is detectable inthe blood of the mammal.
 2. The method of claim 1, where the mammal is ahuman.
 3. The method of claim 2, wherein the defective recombinant Ad2adenovirus is administered by intravenous injection.
 4. The method ofclaim 1, wherein the nucleic acid sequence is positioned in the locationof the deletion of the E1A coding region.
 5. The method of claim 4,wherein the vector further comprises an E3 coding region having adeletion.
 6. The method of claim 4, wherein the nucleic acid sequencecomprises the S gene of hepatitis B virus.
 7. The method of claim 6,wherein the nucleic acid sequence further comprises a pre-S2 gene ofhepatitis B virus, wherein the pre-S2 gene is located immediatelyupstream of the S gene.
 8. The method of claim 2, wherein the vectorfurther comprises an E3 coding region having a deletion.
 9. The methodof claim 8, wherein the nucleic acid sequence is positioned in thelocation of the deletion within the E3 region.
 10. The method of claim9, wherein the nucleic acid sequence comprises the S gene of hepatitis Bvirus.
 11. The method of claim 10, wherein the nucleic acid sequencefurther comprises a pre-S2 gene of hepatitis B virus, wherein the pre-S2gene is located immediately upstream of the S gene.
 12. A method oftransferring a nucleic acid sequence encoding a hepatitis B virus Santigen to a mammal to induce an antibody response in said mammalcomprising: administering a defective recombinant Ad5 adenoviral vectorto the mammal intravenously, wherein the vector comprises: a) an E1Apromoter; b) a deletion of the entire E1A coding region; and c) anucleic acid sequence encoding a hepatitis B virus S antigen operativelylinked to the E1A promoter; wherein the hepatitis B virus S antigen isexpressed in the mammal, thereby inducing an antibody response to thehepatitis B virus S antigen in the mammal, wherein the antibody responseis detectable in the blood of the mammal.
 13. The method of claim 12,where the mammal is a human.
 14. The method of claim 13, wherein thenucleic acid sequence is positioned in the location of the deletion ofthe E1A coding region.
 15. The method of claim 14, wherein the vectorcomprises an E3 coding region having a deletion.
 16. The method of claim14, wherein the nucleic acid sequence comprises the S gene of hepatitisB virus.
 17. The method of claim 16, wherein the nucleic acid sequencefurther comprises a pre-S2 gene of hepatitis B virus, wherein the pre-S2gene is located immediately upstream of the S gene.
 18. The method ofclaim 13, wherein the vector further comprises an E3 coding regionhaving a deletion.
 19. The method of claim 18, wherein the nucleic acidsequence is positioned in the location of the deletion within the E3region.
 20. The method of claim 18, wherein the nucleic acid sequencecomprises the S gene of hepatitis B virus.
 21. The method of claim 20,wherein the nucleic acid sequence further comprises a pre-S2 gene ofhepatitis B virus, wherein the pre-S2 gene is located immediatelyupstream of the S gene.
 22. The method of claim 6, wherein the antibodyresponse comprises a serum anti-HBs antibody titer of at least 10mIU/ml.
 23. The method of claim 10, wherein the antibody responsecomprises a serum anti-HBs antibody titer of at least 10 mIU/ml.
 24. Themethod of claim 16, wherein the antibody response comprises a serumanti-HBs antibody titer of at least 10 mIU/ml.